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  • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
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  • A61K38/1816—Erythropoietin [EPO]
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  • A61K38/18—Growth factors; Growth regulators
  • A61K38/185—Nerve growth factor [NGF]; Brain derived neurotrophic factor [BDNF]; Ciliary neurotrophic factor [CNTF]; Glial derived neurotrophic factor [GDNF]; Neurotrophins, e.g. NT-3
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  • A61K38/18—Growth factors; Growth regulators
  • A61K38/1858—Platelet-derived growth factor [PDGF]
  • A61K38/1866—Vascular endothelial growth factor [VEGF]
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  • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • A61K38/19—Cytokines; Lymphokines; Interferons
  • A61K38/196—Thrombopoietin
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  • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
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  • A61K38/27—Growth hormone [GH], i.e. somatotropin
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  • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
  • A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
  • A61L27/38—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells
  • A61L27/3895—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells using specific culture conditions, e.g. stimulating differentiation of stem cells, pulsatile flow conditions
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  • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
  • A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
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  • A61P1/16—Drugs for disorders of the alimentary tract or the digestive system for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
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  • A61P17/00—Drugs for dermatological disorders
  • A61P17/02—Drugs for dermatological disorders for treating wounds, ulcers, burns, scars, keloids, or the like
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  • A61P19/04—Drugs for skeletal disorders for non-specific disorders of the connective tissue
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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  • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
  • A61P7/04—Antihaemorrhagics; Procoagulants; Haemostatic agents; Antifibrinolytic agents
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  • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
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  • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
  • A61L2300/00—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices
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  • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
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  • C12N2501/10—Growth factors
  • C12N2501/13—Nerve growth factor [NGF]; Brain-derived neurotrophic factor [BDNF]; Cilliary neurotrophic factor [CNTF]; Glial-derived neurotrophic factor [GDNF]; Neurotrophins [NT]; Neuregulins
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  • C12N2502/28—Vascular endothelial cells

Definitions

• the present invention relates to the use of at least one growth factor in isolated form for the cultivation of primarily differentiated cells, for the locally specific and/or directed differentiation of adult cells and/or for the regeneration of bones, tissues and/or endocrine organs.
• EGF epidermal growth factor
• VEGF vascular endothelial growth factor
• HGF hepatocyte growth factor
• tissue extracts such as, for example, from the pituitary or the hypothalamus are particularly suitable for bringing about multiplication of hepatocyte cells (see, for example, U.S. Pat. No. 6,008,047).
• Such animal or, occasionally, human extracts have already been added to cell cultures.
• the use of animal or human tissue extracts is, however, problematic in laboratory work or in clinical use owing to transmissible viral diseases such as, for example, BSE, pig or sheep viruses.
• the use of such extracts demonstrates the lack of knowledge about the actually relevant factors and their potential uses and effects.
• a further substantial disadvantage is that through such heterogeneous extracts, which are generally difficult to define and depend considerably on the source used, there is also introduction into the culture of factors which, in some circumstances, bring about unwanted side effects or properties on clinical use. Accurate knowledge of the factors and controlled dosage thereof would therefore be both an important factor for being able to multiply and differentiate cells, especially in the area of tissue engineering, appropriately, and for inducing structural processes of three-dimensional (3-D) regeneration.
• cells such as, for example, hepatocytes are still embedded after the multiplication phases in a gel in order to avoid the formation of further, also large, aggregates (superaggregates).
• These gels are two-dimensional in extent, comprise a high cell density and therefore stop cell multiplication.
• Such 2-D gel inclusions which result in layers, have already been described by Bader et al. (1995), Artif. Organs, 19, 368-374, as sandwich model or gel entrapment.
• the embedding of aggregates in gels results in an improvement in maintenance of differentiation, it does not result in further growth.
• a shape-creating growth from a few precursor cells to a 3-D structure and an inductive behavior for neighborhood processes in the sense of tissue regeneration in vitro and in vivo has not to date been possible.
• inductive growth behavior of cells means a considerable innovation in particular for therapeutic or biotechnological processes.
• Such a growth behavior should, assisted by a 3-D supporting matrix, allow growth not only in the sense of colonization or structural remodeling but in fact be able to allow directed de novo formation from an induction nucleus. Such processes take place in ontogenesis and build upon a pre-existing strom.
• LIF leukemia inhibitory factor
• CNTF ciliary neurotropic factor
• GDNF glial derived neurotrophic factor
• NGF nerve growth factor
• TPO thrombopoietin
• EPO erythropoietin
• GH growth hormone
• LIF leukemia inhibitory factor
• CNTF ciliary neurotropic factor
• the invention therefore relates to a method for multiplying and differentiating cells in vitro, in which the growth process of the cells is initiated and terminated, and structurally guided, by the use of the growth factors TPO and/or EPO and/or GH, especially HGH and/or somatostatin and/or LIF and/or CNTF.
• TPO is also known for example as c-Mpl ligand, mpl ligand, megapoietin or megakariocyte growth and development factor and has to date not been employed in the culturing of, for example, adult hepatocytes or other primary cells apart from platelets and their precursors.
• TPO is essentially necessary for the development and proliferation of megakariocytes and platelets and thus for the formation of blood platelets.
• TPO is constitutively produced in the liver and in the kidneys as 332 amino acid-long protein.
• TGF beta transforming growth factor beta
• GM-CSF granulocyte-macrophage stimulating factor
• GHRH growth hormone releasing hormone
• TRH thyrotropin-releasing hormone
• GnRH gonadotropin-releasing hormone
• CHC corticotropin-releasing hormone
• ADH antidiuretic hormone
• oxytocin prolactin, adrenocorticotropin
• beta-celltropin beta-celltropin
• lutrotropin vasopressin.
• somatostatin and/or TGF beta and/or prostaglandins are also suitable for terminating the growth process of the invention.
• the individual concentrations of the growth factors in solution are normally about 1 to about 100 ng/ml, preferably about 10 to about 50 ng/ml, in particular about 10 to about 20 ng/ml. However, in the case of local coatings, the concentrations of the growth factors may also be a multiple thereof.
• GHRH growth hormone releasing hormone
• TRH thyrotropin-releasing hormone
• GnRH gonadotropin-releasing hormone
• CHC corticotropin-releasing hormone
• somatostatin dopamines, antidiuretic hormone (ADH) and/or oxytocin
• prolactin adrenocorticotropin
• beta-celltropin beta-celltropin
• vasopressin vasopressin
• nerve regeneration factors preferably nerve growth factor (NGF) and/or one or more vessel regeneration factors, preferably vascular endothelial growth factor (VEGF) and/or platelet derived growth factor (PDGF).
• NGF nerve growth factor
• vessel regeneration factors preferably vascular endothelial growth factor (VEGF) and/or platelet derived growth factor (PDGF).
• Said growth factors can generally be purchased commercially but can also be prepared by gene manipulation by methods known to the skilled worker. They include not only the naturally occurring growth factors but also derivatives or variants having substantially the same biological activity.
• TPO can be purchased commercially from CellSystems GmbH, St Katharinen.
• the use of human TPO is preferred for cultivating human adult hepatocytes.
• preparation and characterization of TPO and its variants is described for example in EP 1 201 246, WO 95/21919, WO 95/21920 and WO 95/26746.
• Suitable TPO variants are the TPO derivatives described in WO 95/21919 or the allelic variants or species homologs described in WO 95/21920 or the pegylated TPO described in WO 95/26746 and EP 1 201 246, without restriction thereto.
• Pegylated TPO means for the purposes of the present invention TPO derivatives which are linked to an organic polymer such as, for example, polyethylene glycol, polypropylene glycol or polyoxyalkylene.
• Further variants of TPO also mean derivatives of TPO which have a sequence identity of less than 100% and nevertheless have the activity of TPO, as described preferably in EP 1 201 246.
• TPO derivatives normally have a sequence identity of at least 70%, preferably at least 75%, especially at least 80% and in particular at least 85% compared with human TPO including fragments thereof having TPO activity.
• a particularly preferred TPO activity for the purposes of the present invention is the speeding up of proliferation, differentiation and/or maturation of megakaryocytes or megakaryocyte precursors in platelet-producing forms of these cells by TPO or its variants.
• EPO is also referred to as embryonic form of TPO and is described with its variants for example in EP 0 148 605, EP 0 205 564, EP 0 209 539, EP 0 267 678 or EP 0 411 678.
• growth factor is accordingly not restricted according to the present invention to the naturally occurring forms, but also includes non-naturally occurring forms and variants or derivatives.
• growth factor includes according to the present invention not only growth promoters but also growth inhibitors such as, for example, somatostatin, TGF beta and/or prostaglandins.
• growth inhibitors are particularly suitable for suppressing or inhibiting the growth of mutated cells such as, for example, tumor cells, by highly concentrated local use thereof simultaneously or sequentially, for example also by means of hydrogels or slow-release materials.
• the growth process of the invention is carried out in a culture suitable for the particular cells. It is possible in this connection by means of a suitable device for the cell aggregates formed where appropriate during the growth process to be broken up and, where appropriate, encapsulated and, where appropriate, frozen.
• An example of a suitable device is a grid having, for example, a cutting mesh structure for example 500 ⁇ m in size, which has the effect that new subsidiary aggregates of, for example, hepatocytes can be repeatedly produced. This can advantageously take place in a completely closed system. It is possible in particular to employ contactless, automatically or manually controlled pumping systems which consist for example of piston pumps or generate directed flows generated magnetically or by compressed air compression of tubings. In the presence of endothelial cells it is possible through the shear stress in a perfused bioreactor for spontaneous confluence of the endothelial cells on the surfaces of the aggregates to occur, which may be advantageous for further use.
• Materials suitable for the encapsulation are suitable ones which are known to the skilled worker and in which, for example, structured shapes or spaces are integrated and make an in situ growth structure or enlargement possible.
• An alternative possibility is for the capsule to be dispensed with and, for example, an endothelialization and thus optimal hemocompatibility to be achieved in the presence of endothelial cells.
• the growth process of the cells is locally initiated and terminated, and structurally guided, preferably by a biological matrix.
• the biological matrix is in this case for example treated with one of said growth factors or with a combination of said growth factors as mixture or sequentially. This makes 3-D regeneration and/or artificial guidance of tissue repair or tissue culturing possible even with adult cell systems.
• the biological matrix is normally an implant, e.g. a stent, a patch or a catheter, a transplant, e.g. a skin transplant and/or a supporting material for the growth of cells, e.g. a so-called slow release material, e.g. a hydrogel for example based on fibrin and/or polymers such as, for example, polylactide or polyhydroxy-alkanoate, and/or alginates, a bone substitute material, e.g. tricalcium phosphate, an allogeneic, autologous or xeinogeneic acellularized or non-acellularized tissue, e.g.
• a so-called slow release material e.g. a hydrogel for example based on fibrin and/or polymers such as, for example, polylactide or polyhydroxy-alkanoate, and/or alginates
• a bone substitute material e.g. tricalcium phosphate
• a heart valve venous valve, arterial valve, skin, vessel, aorta, tendon, comea, cartilage, bones, tracea, nerve, miniscus, intervertebral disc, ureters, urethra or bladder (see, for example, EP 0 989 867 or EP 1 172 120), a matrix such as, for example, a laminin, collagen IV and/or Matrigel matrix, preferably a feeder layer such as, for example, collagen I, 3T3 and/or MRC-5 feeder layer, or a collagen fabric.
• a matrix such as, for example, a laminin, collagen IV and/or Matrigel matrix
• a feeder layer such as, for example, collagen I, 3T3 and/or MRC-5 feeder layer, or a collagen fabric.
• the biological matrix is precolonized with cells, preferably tissue-specific cells, precursor cells, bone marrow cells, peripheral blood, adipose tissue and/or fibrous tissue, e.g. with adult precursor cells from the bone marrow, by methods known to the skilled worker. It is possible in this way to achieve anticipation of the in vivo wound-healing process in vitro, and thus a shortened reintegration time takes place after implantation in vivo.
• the cells used according to the present invention are in particular adult cells, i.e. primarily differentiated cells which preferably no longer have an embryonic or fetal phenotype, particularly preferably human adult cells.
• adult progenitor cells tissue-specific cells, preferably osteoblasts, fibroblasts, hepatocytes and/or smooth muscle cells.
• mutated cells such as, for example, tumor cells
• growth inhibitors such as somatostatin, TGF beta and/or prostaglandins.
• hydrogels or slow-release materials which have already been mentioned and which comprise at least one of said growth inhibitors or are supplemented therewith, and are applied locally or in the vicinity of the mutated cells.
• the method of the invention is thus particularly suitable for locally specific and/or directed multiplication, structural growth and subsequent differentiation of adult cells and/or for the regeneration of bones, tissues and/or endocrine organs, e.g. of heart valves, venous valves, arterial valves, skin, vessels, aortas, tendons, comea, cartilage, bones, tracea, nerves, miniscus, intervertebral disc, ureters, urethra or bladders.
• heart valves venous valves, arterial valves, skin, vessels, aortas, tendons, comea, cartilage, bones, tracea, nerves, miniscus, intervertebral disc, ureters, urethra or bladders.
• the method of the invention can also be employed for local administration in vivo by said growth factors being employed either alone or in combination as mixture or sequentially, or in combination with said biological matrices or supporting structures, for example for tissue regeneration, such as, for example, liver regeneration, myocardical regeneration or for wound healing in the region of the skin, e.g. for diabetic ulcers, or gingiva.
• tissue regeneration such as, for example, liver regeneration, myocardical regeneration or for wound healing in the region of the skin, e.g. for diabetic ulcers, or gingiva.
• TPO to be applied in a hydrogel, e.g.
• Said growth factors can thus be administered for example before, during or after a liver resection or removal of liver tissue in order to assist liver regeneration.
• the growth factor(s) can be injected directly into the knee joint. It is thus possible for the growth factor(s) to act via the sinovial fluid directly on the formation of a new cartilage structure.
• the present invention also relates to the use of the growth factors TPO and/or EPO and/or GH and/or somatostatin and/or LIF and/or CNTF for producing a medicament for the treatment of regeneration of bones, cartilage, tissues and/or endocrine organs, e.g.
• parenchymal and/or non-parenchymal organs especially of myocardium, heart valves, venous valves, arterial valves, skin, vessels, aortas, tendons, comea, cartilage, bones, tracea, nerves, miniscus, intervertebral disc, liver, intestinal epithelium, ureters, urethra or bladders, or for the treatment of degenerative disorders and/or for assisting the wound healing process, especially in Crohn's disease, ulcerative colitis and/or in the region of the skin, preferably for diabetic ulcers or gingiva and/or for the treatment of liver disorders, especially of cirrhosis of the liver, hepatitis, acute or chronic liver failure and/or wound healing in the muscle region after sports injuries, muscle disorders, bone injuries, soft tissue injuries and/or for improving wound healing and tissue regeneration, e.g.
• EPO dosage in this case makes neoangiogenesis and subsequent or accompanying tissue regeneration possible.
• TGF beta transforming growth factor beta
• prostaglandins granulocyte-macrophage stimulating factor
• GM-CSF growth hormone releasing hormone
• GHRH growth hormone releasing hormone
• TRH thyrotropin-releasing hormone
• GnRH gonadotropin-releasing hormone
• corticotropin-releasing hormone CH
• dopamine antidiuretic hormone
• ADH antidiuretic hormone
• oxytocin prolactin, adrenocorticotropin
• beta-celltropin beta-celltropin
• PDGF platelet derived growth factor
• a further possibility is for a biological matrix or supporting structure comprising at least one of the growth factors TPO, EPO, GH, especially HGH, somatostatin, LIF and/or CNTF, to be used as inductive substrate for 3-D growth and/or regeneration within a multiplication phase or after a multiplication phase for differentiation or for growth arrest.
• at least one of said growth factors can be applied to a stent in combination with a so-called slow-release material, as described by way of example above.
• the present invention therefore relates further also to a biological matrix or supporting structure comprising at least one of the growth factors thrombopoietin (TPO), erythropoietin (EPO), growth factor (GH), especially human growth hormone (HGH), somatostatin, leukemia inhibitory factor (LIF) and/or ciliary neurotropic factor (CNTF), where the biological matrix or supporting structure in this may also additionally comprise at least one of the growth factors TGF beta, prostaglandin, GM-CSF, GHRH, TRH, GnRH, CRH, dopamine, ADH, oxytocin, prolactin, adrenocorticotropin, beta-celltropin, lutrotropin and/or vasopressin and, where appropriate, additionally one or more nerve regeneration factors, preferably nerve growth factor (NGF) and/or one or more vessel regeneration factors, preferably vascular endothelial growth factor (VEGF) and/or platelet derived growth factor (PDGF).
• the biological matrix or supporting structure of the invention is, for example, an implant, a transplant and/or a supporting material for the growth of cells, the biological matrix or supporting structure possibly being a stent, a catheter, a skin, a hydrogel, a bone substitute material, an allogeneic, autologous or xenogeneic, acellularized or non-acellularized tissue, a synthetic tissue, a feeder layer or a fabric such as, for example, a fabric made of collagen, laminin and/or fibronectin with or without synthetic or other type of basic structure, such as, for example, plastic or a biological matrix. Exemplary embodiments have already been described above.
• the biological matrix or supporting structure is, as already described above in detail, preferably already precolonized with tissue-specific cells, precursor cells, bone marrow cells, peripheral blood, adipose tissue and/or fibrous tissue, or already prepared for in vivo colonization or inductive remodeling in vitro.
• the biological matrix or supporting structure can also be coated with a (bio)polymer layer which comprises at least one of said growth factors.
• a (bio)polymer layer which comprises at least one of said growth factors. Fibrin, plasma, collagen and/or polylactides are suitable for example as (bio)polymer layer.
• the present invention also relates to a method for producing a biological matrix or supporting structure of the invention, in which an optionally activated biological matrix or supporting structure is coated with at least one of the growth factors TPO, EPO, GH, in particular HGH, somatostatin, LIF and/or CNTF, where said matrix or supporting structure can optionally be coated with additionally at least one of the growth factors TGF beta, prostaglandin, GM-CSF, GHRH, TRH, GnRH, CRH, dopamine, ADH, oxytocin, prolactin, adrenocorticotropin, beta-celltropin, lutrotropin and/or vasopressin and, where appropriate, additionally with one or more nerve regeneration factors, preferably NGF and/or one or more vessel regeneration factors, preferably VEGF and/or PDGF.
• the activation of the biological matrix or supporting structure can take place for example by means of plasma ionization, e.g. using hydrogen peroxide, or by means of laser activation.
• biodegradable (bio)polymer layer which comprises said growth factor(s).
• Suitable examples for this purpose are fibrin, plasma, blood, collagen and/or polylactides.
• the biological matrix or supporting structure precolonized in vitro with cells, preferably tissue-specific cells, precursor cells, bone marrow cells, peripheral blood, adipose tissue and/or fibrous tissue.
• the present invention also extends to a device for carrying out the method of the invention, where a perfused bioreactor, especially in the form of a closed system, is preferred.
• a single-phase beta tricalcium phosphate is prepared as granules with a microporosity of, for example, >15 ⁇ m and shaped in a mold of a 3-D defect corresponding to a patient's requirement. This normally takes place in a sintering process.
• the material is subsequently treated by plasma ionization so that activation of the surfaces occurs and the construct is placed in a solution with thrombopoietin, erythropoietin and/or growth hormone (GH) and thus coated in small quantities in a defined way.
• GH growth hormone
• an incubation in a solution without previous surface activation or a coating with a biodegradable (bio)polymer layer comprising these growth factors can take place. It is possible in this case to employ for example fibrin, plasma, collagen and/or polylactides.
• This construct is then either immediately introduced into a defect or precolonized in vitro with tissue-specific cells, precursor cells or bone marrow cells. This achieves anticipation of the in vivo wound-healing process in vitro and thus a shortened reintegration time can take place after implantation in vivo (e.g. after 7 days).
• NOF nerve regeneration
• VEGF vessel regeneration
• Combination with the structure-forming factors and environment concepts is of interest in this connection.
• a biological matrix (allogeneic or autologous heart valve with and without acellularization, a synthetic supporting structure made of plastics which resembles the physiological microenvironment of the cardiovascular target tissue in terms of the chemical composition of the collagens and their spatial arrangement) is precoated with thrombopoietin and erythropoietin as growth factors.
• the material is then treated by plasma ionization (e.g. using hydrogen peroxide, H 2 O 2 ), simultaneously achieving sterilization, so that activation of the surfaces occurs and the construct is placed in a solution with thrombopoietin, erythropoietin and/or growth hormone (GH) and thus coated in small quantities in a defined way.
• plasma ionization e.g. using hydrogen peroxide, H 2 O 2
• H 2 O 2 hydrogen peroxide
• GH growth hormone
• incubation in a solution without previous surface activation or coating with a biodegradable (bio)polymer layer comprising these growth factors is possible. Fibrin, plasma, blood, collagen or polylactides can be employed in this case.
• This construct is then either immediately introduced at the required site (heart valve position, as patch or vessel replacement) or precolonized in vitro with tissue-specific cells, precursor cells or bone marrow cells. This achieves anticipation of the in vivo wound-healing process in vitro and thus a shortened reintegration time can take place after implantation in vivo (e.g. after 7 days).
• a combination with factors of nerve regeneration (NGF) or vessel regeneration (VEGF, PDGF) is possible, but not absolutely necessary.
• Urological constructs can be produced in a corresponding manner.
• a mixed liver cell population from a biopsy or a partial sectate are treated with TPO and/or EPO and/or growth hormone, e.g. HGH in a concentration of 10-50 ng/ml by addition to the medium supernatant.
• the seeding cell density is 10 000 cells/cm.
• the cells are treated with 0.005% collagenase and 0.01% trypsin with the addition of 2 g/l albumin or autologous serum (10-20%) for 5 h.
• the cells are then aspirated off and washed three times in culture medium (Williams B (Williams et al. (1971) Exptl. Cell Res., 69, 106) with 2 g/l albumin and then put for sedimentation in a collagen-coated Petri dish.
• Differentiation of the cells can be achieved by overlayering with an extracellular matrix.
• the cells can be prevented from sedimenting by agitation and come together for the aggregation.
• the cells can be paused in an appropriate device over a grid having a cutting mesh structure 500 ⁇ m in size, so that new subsidiary aggregates can be repeatedly produced. This can take place in a completely closed system.
• contactless pumping systems no squeezing by peristaltic systems but directed flows generated magnetically or by compressed air compression of tubings, or piston pumps—automatic or manual) are employed.
• the cells can then be encapsulated and frozen. Structured shapes and spaces can be integrated in the capsule structure, which makes an in situ growth structure and enlargement possible.
• the capsule can be dispensed with and, through the presence of the endothelial cells in this system and targeted addition of these cells, an endothelialization and thus optimal hemocompatibility can be achieved.
• the shear stress in a perfused bioreactor results in spontaneous confluence of the endothelial cells on the surfaces of the aggregates.
• they can be frozen for example in the bags which are already ideally used for the culture.
• collagen tile or fabrics such as laminin, fibronectin with or without synthetic or another type of basic structure such as, for example, plastic or a biological matrix, or spatially defined structures (tubes for nerves, tendons) correspondingly as above.
• These collagen tile or structures are shaped, coated with TPO, EPO and/or growth hormone (GH) and implanted or precolonized with cells of the target tissue (e.g. tenocytes, neurons).
• a biological matrix (allogeneic or autologous heart valve with and without acellularization, a synthetic supporting structure made of plastics which resembles the physiological microenvironment of the target tissue in terms of the chemical composition of the collagens and their spatial arrangement) is precoated with thrombopoietin and erythropoietin as growth factors.
• the material is then treated by plasma ionization (e.g. using hydrogen peroxide, H 2 O 2 ), simultaneously achieving sterilization, so that activation of the surfaces occurs and the construct is placed in a solution with thrombopoietin, erythropoietin and/or growth hormone and thus coated in small quantities in a defined way.
• plasma ionization e.g. using hydrogen peroxide, H 2 O 2
• incubation in a solution without previous surface activation or coating with a biodegradable (bio)polymer layer comprising these growth factors is possible. Fibrin, plasma, collagen and/or polylactides can be employed in this case.
• This construct is then either immediately introduced at the required site (abdominal wall, myocardium, skeletal muscle as patch) or precolonized in vitro with tissue-specific cells, precursor cells or bone marrow cells. This achieves anticipation of the in vivo wound-healing process in vitro and thus a shortened reintegration time can take place after implantation in vivo (e.g. after 7 days).
• a combination with factors of nerve regeneration (NGF) or vessel regeneration (VEGF, PDGF) is possible, but not absolutely necessary.
• EPO is administered systemically and/or topically to the patient by application to the resection surface in conjunction with a polymer.
• the polymer may be a biopolymer such as, for example, fibrin (from, for example, fibrin glue), polymerized plasma, polymerized blood or bioadhesives, e.g. mussel adhesive. However, it may also be synthetic or biological gels or hydrogels.
• the EPO can also be introduced into fabrics which serve to stop bleeding (e.g. collagen fabrics, tamponade, wovens and knits).
• EPO can also be employed for regenerating the liver in chronic liver disorders such as, for example, cirrhosis, fibrosis, hepatitis. It is thus possible for the first time to achieve a therapeutic effect in relation to the liver parenchyma.
• Topical dosage may take place by slow release capsules in the intestinal region or by giving suppositories with gels or local installation with solutions.
• Absorption in the regional vascular area can be optimized by giving pegylated (PEG) compounds, so that systemic effect and thus initiation of the wound-healing process can take place via the regional dosage in the area of inflammation.
• PEG pegylated
• anemia is to be regarded as a prognostic positive factor for patients with Crohn's disease. It was assumed in the past that the anemia is an independent concomitant disorder or is attributable to the wasting due to absorption problems. Our results show that the impairment of wound healing involves a deficiency of endogenous EPO. It is thus possible to treat Crohn's disease very selectively by exogenous dosage of EPO. Further uses are to be found also in the area of ulcerative colitis.
• EPO e.g. collagen fabric
• EPO can be given in a similar manner for all other wound healing requirements, e.g. in the muscle region after sports injuries, muscle disorders, bone injuries, soft-tissue injuries and generally for improving wound healing and tissue regeneration, e.g. after operations, acute and chronic disorders.

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